MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in regulating mRNA stability and translational repression. There is increasing evidence that miRs can modulate gene expression in the cardiovascular system. Endothelial cells (ECs) lining the vascular lumen are sensitive to mechanical factors such as fluid shear stress. During the past two decades, this research team has worked on the mechanisms of mechanotransduction in ECs and the consequent gene expression. The results from them and others indicate that steady and pulsatile shear stresses (PS) with a net forward direction are anti-atherogenic by inducing genes involved in anti-proliferation and anti-inflammation. In contrast, oscillatory shear stress (OS) without a significant forward direction is pro-atherogenic by activating pro-proiferative and pro-inflammatory genes. Based on new evidence in the literature and our recent findings that miRs play an important role in regulating EC genes, we hypothesize that anti-atherogenic (PS) and pro-atherogenic (OS) flow patterns induce distinct patterns of miRs, and hence the differential gene expressions and functional consequences. We will use in vitro, in vivo, and in silico approaches to develop an integrated system to elucidate the roles of miRs in regulating EC functions in response to different flow patterns. This multi-P.I. research project, by combining experimental data obtained from cultured ECs and mouse models with molecular, genomics and systems approaches, will elucidate the mechanisms of functional regulation by miRs in ECs under flows. In order to test our hypothesis, we propose the following five specific aims: (1) to establish miR expression profiles in cultured ECs in response to PS vs. OS. (2) To determine the target mRNAs of miRs in response to PS vs. OS. (3) To decipher the functional gene expression profiles regulated by miRs under PS vs. OS. (4) To elucidate the functional consequences of miR regulation under PS vs. OS. (5) To verify the role of miRs in functional regulation of vascular ECs exposed to different flow patterns in vivo. In this proposal the role of miR in regulating vascular functions will be studied under different flow patterns with a combination of experimental and computational approaches to perform multi-scale analyses from miRs/mRNAs to cellular functions. This innovative, multidisciplinary project includes (a) comprehensive genome-wide approaches to establish the miR profiles in ECs, (b) CLIP-seq approaches to elucidate the interactions between miRs and target mRNAs, (c) systems biology approaches to map the functional gene expression and biological consequence regulated by miRs, and (d) in vivo approaches in lesion-induction mice to validate the roles of miRs under different flow patterns established in vitro. The results will enhance the mechanistic insights of the roles of mechano- regulation and functional genomics at the systems biology level and may contribute to the development of novel approaches for the diagnosis and treatment of cardiovascular diseases. PUBLIC HEALTH RELEVANCE: MicroRNAs (miRs) are small non-coding RNAs that play crucial roles in the regulation of mRNA stability and translational repression, leading to the modulation of ~30% of the human genome. We will use in vitro, in vivo, and in silico technologies to develop an integrated systems approach to elucidate the roles of miRs in regulating endothelial functions in normal and pathophysiological flow conditions. The result may and may contribute to the development of novel approaches for the diagnosis and treatment of cardiovascular diseases.